The role of Ala231 and Trp227 in the substrate specificities of fungal 17尾-hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase: Steroids versus smaller substrates

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• 作者：Mojca Brunskole &Scaron ; vegelj ; Jure Stojan ; Tea Lani&scaron ; nik Ri啪ner ; ^{Tea.Lanisnik-Rizner@mf.uni-lj.si}
• 关键词：3HNR ; trihydroxynaphthalene reductase ; 17尾-HSDcl ; 17尾-hydroxysteroid dehydrogenase from fungus Cochliobolus lunatus ; DBO ; 4 ; 5-dihydroxy-2H-benzopyran-2-one ; DDBO ; 2 ; 3-dihydro-2 ; 5-dihydroxy-4H-benzopyran-4-one ; PQ ; 9 ; 10-phenanthrenequinone ; TLC ; thin-lay
• 刊名：The Journal of Steroid Biochemistry and Molecular Biology
• 出版年：2012
• 期刊代码：172_09600760
• 类别：med
• 出版时间：March, 2012
• 卷：129
• 期：1-2
• 页码：92-98
• 文件大小：927 K

摘要

17尾-Hydroxysteroid dehydrogenase and trihydroxynaphthalene reductase from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus; 17尾-HSDcl and 3HNR, respectively) are two homologous short-chain dehydrogenase/reductase proteins that are 58%identical and have 86%similar amino acids. The minor differences in their substrate-binding regions are believed to be crucial for their substrate specificities. 3HNR shows high affinity for substrates with two rings, like trihydroxynaphthalene and 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), while 17尾-HSDcl can accommodate ligands with four rings, like steroids. In the present study, we examined the role of Ala231 in 17尾-HSDcl and Trp227 in 3HNR, as the potential key amino acids in the determination of substrate recognition based on size. We constructed Ala231Trp 17尾-HSDcl and Trp227Ala 3HNR mutant proteins and used spectrophotometric analyses to compare their catalytic activities with those of the wild-type enzymes, for oxidation of 4-estrene-17尾-ol-3-one and DDBO and for reduction of 4-estrene-3,17-dione and 9,10-phenanthrenequinone (PQ). The Ala231Trp side-chain substitution in 17尾-HSDcl abolished and decreased (by 14.6-fold) the initial rates for steroid oxidation and reduction, respectively, while the initial rate for PQ reduction was increased 5.6-fold. The bulky Trp227Ala side-chain substitution in 3HNR enabled oxidation of 4-estrene-17尾-ol-3-one, increased the initial rates for reduction of 4-estrene-3,17-dione and PQ by 4.5-fold and 1.5-fold, respectively, while the initial rate for DDBO oxidation was decreased 4.1-fold. Our TLC analysis and docking simulations also support these findings. Our study thus confirms the important roles of Ala231 in 17尾-HSDcl and Trp227 in 3HNR, for the selection between larger and smaller substrates.

Article from a special issue on steroids and microorganisms.